scholarly journals Efficient formation of bipolar microtubule bundles requires microtubule-bound γ-tubulin complexes

2005 ◽  
Vol 169 (2) ◽  
pp. 297-308 ◽  
Author(s):  
Marcel E. Janson ◽  
Thanuja Gangi Setty ◽  
Anne Paoletti ◽  
P.T. Tran
Keyword(s):  
Epithelial Cells ◽  
Fission Yeast ◽  
Linear Array ◽  
Spatial Separation ◽  
Wild Type ◽  
Basic Form ◽  
Yeast Protein ◽  
Polarized Epithelial Cells ◽  
In Cells

The mechanism for forming linear microtubule (MT) arrays in cells such as neurons, polarized epithelial cells, and myotubes is not well understood. A simpler bipolar linear array is the fission yeast interphase MT bundle, which in its basic form contains two MTs that are bundled at their minus ends. Here, we characterize mto2p as a novel fission yeast protein required for MT nucleation from noncentrosomal γ-tubulin complexes (γ-TuCs). In interphase mto2Δ cells, MT nucleation was strongly inhibited, and MT bundling occurred infrequently and only when two MTs met by chance in the cytoplasm. In wild-type 2, we observed MT nucleation from γ-TuCs bound along the length of existing MTs. We propose a model on how these nucleation events can more efficiently drive the formation of bipolar MT bundles in interphase. Key to the model is our observation of selective antiparallel binding of MTs, which can both explain the generation and spatial separation of multiple bipolar bundles.

scholarly journals Ezrin binding domain-deficient NHERF attenuates cAMP-mediated inhibition of Na+/H+ exchange in OK cells

2001 ◽  
Vol 281 (2) ◽  
pp. F374-F380 ◽  
Author(s):  
Edward J. Weinman ◽  
Deborah Steplock ◽  
James B. Wade ◽  
Shirish Shenolikar
Keyword(s):  
Epithelial Cells ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Proposed Model ◽  
Exchange Activity ◽  
In Cells

Na+/H+ exchanger regulatory factor (NHERF), an essential protein cofactor in cAMP-mediated inhibition of Na+/H+ exchange transporter 3 (NHE3), facilitates the formation of a signal complex of proteins that includes NHE3, NHERF, and ezrin. This model for NHE3 regulation was developed in fibroblasts and its applicability to epithelial cells remains to be established. Opossum kidney (OK) cells were transfected with either empty vector (control), full-length mouse (m) NHERF(1–355), or a truncated mNHERF(1–325) that lacked ezrin binding and had been demonstrated in fibroblasts to bind NHE3 but not mediate its cAMP-associated inhibition. 8-Bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) at 10−4 M inhibited Na+/H+ exchange activity in control and OK cells expressing wild-type mNHERF(1–355) by >60% but by <10% in cells expressing mNHERF(1–325). NHE3 coimmunoprecipitated with mNHERF(1–325), but cAMP phosphorylation of NHE3 was impaired in cells expressing mNHERF(1–325). The inhibitory effect of hyperosmolality on NHE3 activity and the uptake of 3- O-methyl-d-glucose was the same in all three cell lines. Cell surface expression of NHE3 was not changed by cAMP in any of the cells lines. These data indicate that disruption of the NHERF-ezrin signal complex attenuates the inhibitory effect of cAMP on NHE3 activity in OK cells and provides evidence supporting the proposed model of protein kinase A regulation of NHE3 in epithelial cells.

The ΔF508 mutation shortens the biochemical half-life of plasma membrane CFTR in polarized epithelial cells

2001 ◽  
Vol 280 (1) ◽  
pp. C166-C174 ◽  
Author(s):  
Ghanshyam D. Heda ◽  
Mridul Tanwani ◽  
Christopher R. Marino
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Half Life ◽  
Immunoblot Analysis ◽  
Wild Type ◽  
Δf508 Cftr ◽  
Polarized Epithelial Cells ◽  
Physical And Chemical ◽  
Biochemical Stability

Although the biosynthetic arrest of the ΔF508 mutant of cystic fibrosis transmembrane conductance regulator (CFTR) can be partially reversed by physical and chemical means, recent evidence suggests that the functional stability of the mutant protein after reaching the cell surface is compromised. To understand the molecular basis for this observation, the current study directly measured the half-life of ΔF508 and wild-type CFTR at the cell surface of transfected LLC-PK1 cells. Plasma membrane CFTR expression over time was characterized biochemically and functionally in these polarized epithelial cells. Surface biotinylation, streptavidin extraction, and quantitative immunoblot analysis determined the biochemical half-life of plasma membrane ΔF508 CFTR to be ∼4 h, whereas the plasma membrane half-life of wild-type CFTR exceeded 48 h. This difference in biochemical stability correlated with CFTR-mediated transport function. These findings indicate that the ΔF508 mutation decreases the biochemical stability of CFTR at the cell surface. We conclude that the ΔF508 mutation triggers more rapid internalization of CFTR and/or its preferential sorting to a pathway of rapid degradation.

scholarly journals Intramitotic controls in the fission yeast Schizosaccharomyces pombe: the effect of cell size on spindle length and the timing of mitotic events.

1990 ◽  
Vol 110 (5) ◽  
pp. 1617-1621 ◽  
Author(s):  
I M Hagan ◽  
P N Riddle ◽  
J S Hyams
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Average Length ◽  
Nuclear Migration ◽  
Yeast Cells ◽  
Reverse Direction ◽  
Wild Type ◽  
Spindle Elongation ◽  
Anaphase B ◽  
In Cells

We have used a new cinemicroscopy technique in combination with antitubulin immunofluorescence microscopy to investigate the timing of mitotic events in cells of the fission yeast Schizosaccharomyces pombe having lengths at division between 7 and 60 microns. Wild-type fission yeast cells divide at a length of 14 microns. Separation of daughter nuclei (anaphase B) proceeds at a rate of 1.6 +/- 0.2 microns min-1, until the spindle extends the length of the cell. Coincident with spindle depolymerization, the nuclei reverse direction and take up positions that will become the center of the two daughter cells. This post-mitotic nuclear migration occurs at a rate of 1.4 +/- 0.5 microns-1. In cells in which the weel+ gene is overexpressed fivefold and that have an average length at mitosis of 28 microns, the rate of nuclear separation was only slightly reduced but, as spindles in these cells measure 20-22 microns, the duration of anaphase B was extended by approximately 40%. By contrast, in the mutant weel.50, which divides at 7 microns, both the rate and duration of anaphase B were indistinguishable from wild type. Nuclei reach the ends of these cells earlier but remain there until a point corresponding to the time of postmitotic nuclear migration in wild type. Thus, the events of mitosis can be extended but not abbreviated. These results are discussed in terms of a mitotic termination control that monitors many different events, one of which is spindle elongation.

scholarly journals cis Effects in Adeno-Associated Virus Type 2 Replication

2007 ◽  
Vol 81 (18) ◽  
pp. 9976-9989 ◽  
Author(s):  
Peter Ward ◽  
Nathalie Clément ◽  
R. Michael Linden
Keyword(s):  
Dna Replication ◽  
Wild Type Virus ◽  
Wild Type ◽  
Packaging Signal ◽  
Helper Plasmid ◽  
Adeno Associated Virus ◽  
Plasmid Transfection ◽  
High Yields ◽  
In Cells

ABSTRACT We have utilized deletion mutants of adeno-associated virus (AAV) to investigate which elements of the AAV genome are required in cis for high yields of the wild-type virus in a plasmid transfection assay and in addition whether these elements affect primarily AAV DNA replication or encapsidation. All tested deletions from within the Rep region demonstrated a modest, approximately threefold, decrease in viral production. Deletions within the cap region resulted in markedly less virus. Previous observations suggested that in cells in which recombinant AAV (rAAV) was produced, as in our assay with the helper plasmid pDG, there is a substantial excess of empty capsids. Cotransfections of high- and low-yielding constructs demonstrated that under conditions where Cap is abundant, the constructs with cap deletions did not package efficiently. These observation suggest that the lower yields of rAAV cannot be entirely due to lack of capsids but that elements within the cap region of the wild-type genome are important for efficient encapsidation. The production of virus by the mutants we tested was, however, not consistent with the disruption of a cis-acting packaging signal. Apparently, when Cap is provided “in trans,” encapsidation is inefficient. A second observation is that there were equivalent amounts of replicated but unencapsidated viral DNA in cells transfected with each of our constructs. We propose that, in accord with the previously proposed link between DNA replication and encapsidation, the total amount of AAV DNA replication can be limited by the efficiency of encapsidation.

Filamentous particle formation by human parainfluenza virus type 2

Microbiology ◽  
2000 ◽  
Vol 81 (5) ◽  
pp. 1305-1312 ◽  
Author(s):  
Qizhi Yao ◽  
Richard W. Compans
Keyword(s):  
Epithelial Cells ◽  
Virus Type ◽  
Particle Formation ◽  
Parainfluenza Virus ◽  
Morphological Properties ◽  
Dependent Manner ◽  
Polarized Epithelial Cells ◽  
Human Parainfluenza Virus ◽  
Filamentous Particle

Some paramyxoviruses form long filamentous virus particles: however, the determinants of filament formation and the role of such particles in virus transmission and pathogenicity are not clearly defined. By using conventional immunofluorescence microscopy, we found that human parainfluenza virus type 2 (HPIV2) forms filamentous particles ranging from 5 to 15 μm in length in virus-infected, polarized epithelial cells. The formation of filamentous particles was found to be virus type-specific and was not observed when the same cell types were infected with parainfluenza virus type 3 or Sendai virus, suggesting that different paramyxovirus genera exhibit distinct morphological properties. HPIV2 filamentous particle formation was found to be inhibited by cytochalasin D (CD) or jasplakinolide treatment in a dose-dependent manner. In the presence of 4 μg/ml CD or 1 μM jasplakinolide, the formation of filamentous particles was completely abolished, although similar haemagglutination and p.f.u. titres of virus were found to be released into the culture medium at 24 h post-infection. These observations indicate that host cell components, including the actin microfilament network, are important determinants of the morphology of parainfluenza viruses. The predominance of filamentous particles in polarized epithelial cells may reflect specific pathogenic roles of these particles in infection of human epithelial tissues.

Umatilla Virus Association with Fibrils and Tubules in Cultured Cells

1980 ◽  
Vol 38 ◽  
pp. 476-477
Author(s):  
Neil M. Foster ◽  
Ruth D. Breckon
Keyword(s):  
Bluetongue Virus ◽  
Linear Array ◽  
Cultured Cells ◽  
Intimate Association ◽  
Infected Cells ◽  
Dark Staining ◽  
In Cells

Macrotubules have been described1 in cells infected with Umatilla virus (UMAV), an orbivirus for which bluetongue virus (BTV) is the protype. Macrotubules, often in linear array, were observed in the cytoplasm and in intimate association with viroplasms of infected cells. Macrotubules had outside and inside diameters of 20 and 15 nm and many had dark-staining centers with diameters similar to the interiors of the tubules. UMAV was 60 nm and the RNA core was 30 nm in diameter. This report describes the association of UMAV with macrotubules and two types of microtubules.

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